The library preparation strategies for generating host genomic (HG) and microbial metagenomic (MG) data are generally the same.
Short-read sequencing libraries requires DNA to be sheared to the desired fragment-length (usually 400-500 nucleotides), which can be achieved using either chemical (e.g., restriction enzymes) or physical (e.g. ultrasonication) procedures. Some long-read sequencing libraries intend to keep the largest DNA molecules possible, although some others recommend fragmenting to optimal mid-length molecules (e.g., around 10,000 nucleotides for Pacbio HiFi). After fragmentation, many library preparation protocols require repairing molecule ends by converting 5’-protruding and/or 3’-protruding ends to 5’-phosphorylated, blunt-end (see below) molecules.
In shotgun libraries adaptors are merged to DNA template molecules through chemical ligation (e.g., using a ligase enzyme). The ligation process is slightly different depending on whether the DNA template has blunt- or sticky-ends. In blunt ends, both strands are of equal length – i.e. they end at the same base position, leaving no unpaired bases on either strand, while in sticky ends, one strand is longer than the other. Some protocols deliberately create sticky-ends from blunt-end fragmented DNA molecules by adding a single adenine base to form an overhang by an A-tailing reaction. This A overhang allows adapters containing a single thymine overhanging base to pair with the DNA fragments.
An example of a blunt-end molecule:
An example of a sticky-end molecule:
|Blunt-End Single-Tube (BEST) library prep protocol
|Santa Cruz Reaction (SCR) single-stranded library prep protocol
|SMRTbell prep kit 3.0 for PacBio HiFi Sequencing